Protein synthesis is directed by nucleic acids through the intermediacy of messenger RNA (mRNA). Antisense methodology is the complementary hybridization of relatively short oligonucleotides to mRNA or DNA such that the normal, essential functions, such as protein synthesis, of these intracellular nucleic acids are disrupted. Hybridization is the sequence-specific hydrogen bonding via Watson-Crick base pairs of oligonucleotides to RNA or single-stranded DNA. Such base pairs are said to be complementary to one another.
The naturally occurring events that provide the disruption of the nucleic acid function, discussed by Cohen in Oligonucleotides: Antisense Inhibitors of Gene Expression, CRC Press, Inc., Boca Raton, Fla. (1989) are thought to be of two types. The first, hybridization arrest, describes the terminating event in which the oligonucleotide inhibitor binds to the target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid. Methyl phosphonate oligonucleotides: Miller, P. S. and Ts'O, P. O. P. (1987) Anti-Cancer Drug Design, 2:117-128, and α-anomer oligonucleotides are the two most extensively studied antisense agents which are thought to disrupt nucleic acid function by hybridization arrest.
Another means by which antisense oligonucleotides disrupt nucleic acid function is by hybridization to a target mRNA, followed by enzymatic cleavage of the targeted RNA by intracellular RNase H. A 2′-deoxyribofuranosyl oligonucleotide or oligonucleotide analog hybridizes with the targeted RNA and this duplex activates the RNase H enzyme to cleave the RNA strand, thus destroying the normal function of the RNA. Phosphorothioate oligonucleotides are the most prominent example of an antisense agent that operates by this type of antisense terminating event.
Considerable research is being directed to the application of oligonucleotides and oligonucleotide analogs as antisense agents for diagnostics, research applications and potential therapeutic purposes. At least for therapeutic purposes, the antisense oligonucleotides and oligonucleotide analogs must be transported across cell membranes or taken up by cells to exhibit their activity. However, due to the large size and unfavorable charge-size ratio of oligonucleotides, their cellular uptake is very limited. Numerous efforts have focused on increasing this membrane permeability and cellular delivery of oligonucleotides.
Efforts aimed at improving the transmembrane delivery of nucleic acids and oligonucleotides have utilized protein carriers, antibody carriers, liposomal delivery systems, electroporation, direct injection, cell fusion, viral vectors, and calcium phosphate mediated transformation. However, many of these techniques are limited by the types of cells in which transmembrane transport is enabled and by the conditions needed for achieving such transport. An alternative that is particularly attractive for the transmembrane delivery of oligonucleotides is the modification of the physico-chemical properties of oligonucleotides via conjugation to a molecule that facilitates transport.
One method for increasing membrane or cellular transport of oligonucleotides is the attachment of a pendant lipophilic group. Ramirez, F., Mandal, S. B. and Marecek, J. F., (1982) J. Am. Chem. Soc., 104:5483, introduced the phospholipid group 5′-O-(1,2-di-O-myristoyl-sn-glycero-3-phosphoryl) into the dimer TpT independently at the 3′ and 5′ positions. Subsequently Shea, R. G., Marsters, J. C. and Bischofberger, N. (1990), Nuc. Acids Res., 18:3777, disclosed oligonucleotides having a 1,2-di-O-hexyldecyl-rac-glycerol group linked to a 5′-phosphate on the 5′-terminus of the oligonucleotide. Certain of the Shea et. al. authors also disclosed these and other compounds in patent application PCT/US90/01002. A further glucosyl phospholipid was disclosed by Guerra, F. I., Neumann, J. M. and Hynh-Dinh, T. (1987), Tetrahedron Letters, 28:3581.
In other work, a cholesteryl group was attached to the inter-nucleotide linkage between the first and second nucleotides (from the 3′ terminus) of an oligonucleotide. This work is disclosed in U.S. Pat. No. 4,958,013 and further in Letsinger, R. L., Zhang, G., Sun, D. K., Ikeuchi, T. and Sarin, P. S. (1989), Proc. Natl. Acad. Sci. USA, 86:6553. Additional approaches to the delivery and study of oligonucleotides have involved the conjugation of a variety of other molecules and reporter groups. The aromatic intercalating agent anthraquinone was attached to the 2′ position of a sugar fragment of an oligonucleotide as reported by Yamana, K., Nishijima, Y., Ikeda, T., Gokota, T. Ozaki, H., Nakano, H., Sangen, O. and Shimidze, T. (1990) Bioconjugate Chem., 1:319; Lemairte, M., Bayard, B. and Lebleu, B. (1986), Proc. Natl. Acad. Sci. USA, 84:648; and Leonetti, J.-P., Degols, G. and Lebleu, B. (1990), Bioconjugate Chem., 1:149. Lysine and polylysines have also been conjugated to oligonucleotides to improve their charge-size characteristics. The poly(L-lysine) was linked to the oligonucleotide via periodate oxidation of the 3′-terminal ribose followed by reduction and coupling through a N-morpholine ring. Oligonucleotide-poly(L-lysine) conjugates are described in European Patent application 87109348.0. In this instance the lysine residue was coupled to a 5′ or 3′ phosphate of the 5′ or 3′ terminal nucleotide of the oligonucleotide. A disulfide linkage has also been utilized at the 3′ terminus of an oligonucleotide to link a peptide to the oligonucleotide as is described by Corey, D. R. and Schultz, P. G. (1987), Science, 238:1401; Zuckermann, R. N., Corey, D. R., and Schultz, P. G. (1988), J. Am. Chem. Soc., 110:1614; and Corey, D. R., Pei, D. and Schultz, P. G. (1989), J. Am. Chem. Soc., 111:8524.
Nelson, P. S., Frye, R. A. and Liu, E. (1989), Nuc. Acids Res., 17:7187 describe a linking reagent for attaching biotin to the 3′-terminus of an oligonucleotide. This reagent, N-Fmoc-O-DMT-3-amino-1,2-propanediol is now commercially available from Clontech Laboratories (Palo Alto, Calif.) under the name 3′-Amine on. It is also commercially available under the name 3′-Amino-Modifier reagent from Glen Research Corporation (Sterling, Va.). This reagent was also utilized to link a peptide to an oligonucleotide as reported by Judy, C. D., Richardson, C. D. and Brousseau, R. (1991), Tetrahedron Letters, 32:879. A similar commercial reagent (actually a series of such linkers having various lengths of polymethylene connectors) for linking to the 5′-terminus of an oligonucleotide is 5′-Amino-Modifier C6. These reagents are available from Glen Research Corporation (Sterling, Va.). These compounds or similar ones were utilized by Krieg, A. M., Gmelig-Meyling, F., Gourley, M. F., Kisch, W. J., Chrisey, L. A. and Steinberg, A. D. (1991), Antisense Research and Development, 1:161 to link fluorescein to the 5′-terminus of an oligonucleotide. Other compounds of interest have also been linked to the 3′-terminus of an oligonucleotide. Asseline, U., Delaure, M., Lancelot, G., Toulme, F., Thuong, N. T., Montenay-Garestier, T. and Helene, C. (1984), Proc. Natl. Acad. Sci. USA, 81:3297 described linking acridine on the 3′-terminal phosphate group of an poly (Tp) oligonucleotide via a polymethylene linkage. Haralambidis, J., Duncan, L. and Tregear, G. W. (1987), Tetrahedron Letters, 28:5199 report building a peptide on a solid state support and then linking an oligonucleotide to that peptide via the 3′ hydroxyl group of the 3′ terminal nucleotide of the oligonucleotide. Chollet, A. (1990), Nucleosides & Nucleotides, 9:957 attached an Aminolink 2 (Applied Biosystems, Foster City, Calif.) to the 5′ terminal phosphate of an oligonucleotide. They then used the bifunctional linking group SMPB (Pierce Chemical Co., Rockford, Ill.) to link an interleukin protein to the oligonucleotide.
Conjugation of lipids, reporters, peptides and other molecules to oligonucleotides is not limited to the terminal 3′ and 5′-positions. A wide variety of conjugates have also been reported in the literature wherein attachment is performed at any one or more of the 2′-positions on the nucleotide building blocks of the oligonucleotide. Further conjugates have also been reported wherein attachment occurs on the internucleotide linkage or on one of the atoms of the nucleobase of any one of the nucleotide units of the oligonucleotide. For example, an EDTA iron complex has been linked to the 5 position of a pyrimidine nucleoside as reported by Dreyer, G. B. and Dervan, P. B. (1985), Proc. Natl. Acad. Sci. USA, 82:968. Fluorescein has been linked to an oligonucleotide in the same manner as reported by Haralambidis, J., Chai, M. and Tregear, G. W. (1987), Nucleic Acid Research, 15:4857 and biotin in the same manner as described in PCT application PCT/US/02198. Fluorescein, biotin and pyrene were also linked in the same manner as reported by Telser, J., Cruickshank, K. A., Morrision, L. E. and Netzel, T. L. (1989), J. Am. Chem. Soc., 111:6966. A commercial reagent, Amino-Modifier-dT, from Glen Research Corporation (Sterling, Va.) can be utilized to introduce pyrimidine nucleotides bearing similar linking groups into oligonucleotides.
Manoharan et al. PCT Application WO 93/07883, have also reported the conjugation of oligonucleotides with a variety of molecules such as steroids, reporter molecules, reporter enzymes, vitamins, non-aromatic lipophilic molecules, chelators, porphyrins, intercalators, peptides and proteins through the intermediacy of varied linking groups, such as w-aminoalkoxy and w-aminoalkylamino groups. Conjugation has been reported at the 3′-, 5′-, 2′-, internucleotide linkage and nucleobase positions of oligonucleotides. Such oligonucleotide conjugates are expected to have improved physicochemical properties that facilitated their uptake and delivery into cells as demonstrated by in vitro experiments.
The intracellular and intranuclear delivery of nucleic acids and oligonucleotides, however, is still a challenge. Most often, penetration of heretofore reported oligonucleotide conjugates has been found to be limited. This has typically been a problem because such conjugates have generally been designed to improve the passive absorption of the oligonucleotides where the size, physico-chemical properties and extra-cellular concentration of the conjugate play important limiting roles. This coupled with the limited extra-cellular stability of nucleic acids and oligonucleotides demands the development of novel conjugates that will deliver higher levels of nucleic acids and oligonucleotides into specific tissues and targeted cells. One such approach for delivering oligonucleotides, is to exploit the active transport mechanism of receptor mediated endocytosis.
Unlike many of the methods mentioned above, receptor mediated endocytotic activity can be used successfully both in vitro and in vivo. This mechanism of uptake involves the movement of ligands bound to membrane receptors into the interior of an area that is enveloped by the membrane via invagination of the membrane structure. This process is initiated via activation of a cell-surface or membrane receptor following binding of a specific ligand to the receptor. Many receptor-mediated endocytotic systems are known and have been studied, including those that recognize sugars such as galactose, mannose, mannose-6-phosphate, peptides and proteins such as transferrin, asialoglycoprotein, vitamin B12, insulin and epidermal growth factor (EGF). Receptor mediated endocytosis has been well studied and is known to be a critical pathway for the uptake and internalization of a variety of cellular nutrients. These are highly developed mechanisms because of their critical role in providing nutrients to cells and in maintaining cellular physiology. Thus many examples of the utilization of receptor mediated endocytosis pathways for the delivery of drugs, proteins, nucleic acids and other molecules to cells are known. One way in which this has been applied is the conjugation of essential nutrients that are actively transported into cells with the drug or molecule of interest. The transporters or receptors involved in the uptake are capable of recognizing the nutrient portion of the conjugate and ferrying the entire conjugate into the cell. Examples of nutrients that are actively transported into cells and that may be of use in conjugates include, but are not limited to, folic acid, vitamin B6, cholesterol and vitamin B12. Such molecules have been conjugated to macromolecules such as nucleic acids and oligonucleotides to afford conjugates that exhibit improved cellular penetration. Manorharan et al., PCT Application WO 93/07883; Low et al., U.S. Pat. No. 5,108,921, U.S. Pat. No. 5,416,016.
Many vitamins possess an acid or alcohol functionality that is readily modified for conjugation to oligonucleotides. For example, conjugation of an N-hydroxy succinimide ester of an acid moiety of retinoic acid to an amine function on a linker pendant to an oliogonucleotide resulted in a oligonucleotide-Vitamin A conjugate attached via an amide bond. Retinol has been converted to its phosphoramidite and conjugated to the 5′-terminus of oligonucleotides via a phosphodiester or phosphorothioate linkage. Likewise, vitamin E and vitamin B6 may also be conjugated to oligonucleotides to improve transport into cells.
Pyridoxal (vitamin B6) has specific B6-binding proteins. The role of these proteins in pyridoxal transport has been studied by Zhang and McCormick, Proc. Natl. Acad. Sci. USA, 1991 88, 10407. Zhang and McCormick also have shown that a series of N-(4′-pyridoxyl)amines, in which several synthetic amines were conjugated at the 4′-position of pyridoxal, are able to enter cells by a process facilitated by the B6 transporter. They also demonstrated the release of these synthetic amines within the cell. Other pyridoxal family members include pyridoxine, pyridoxamine, pyridoxal phosphate, and pyridoxic acid. Pyridoxic acid, niacin, pantothenic acid, biotin, folic acid and ascorbic acid can be conjugated to oligonucleotides using N-hydroxysuccinimide esters that are reactive with aminolinkers located on the oligonucleotide, as described above for retinoic acid.
Folic acid and its various forms, such as dihydrofolate and tetrahydrofolate, are essential vitamins that are crucial for the biosynthesis of nucleic acids and therefore are critical to the survival and proliferation of cells. Folate cofactors play an important role in the one-carbon transfers that are critical for the biosynthesis of pyrimidine nucleosides. Cells therefore have a sophisticated system of transporting folates into the cytoplasm. Uptake of folates occurs by two different pathways depending on the cell type. Cells expressing a carrier or transporter for folate that exhibits a low affinity (Kd˜10−6 M) for the vitamin prefer reduced over oxidized forms of folate. Cells that express membrane receptors called folate binding protein (FBP), in contrast, exhibit high binding affinity (Kd˜10−9 M) and prefer the oxidized form of the vitamin. This latter receptor is believed to mediate the uptake of folates into the cytoplasm via endocytosis.
The use of biotin conjugates and also folic acid conjugates to enhance transmembrane transport of exogenous molecules, including oligonucleotides, has been reported by Low et al., U.S. Pat. No. 5,108,921; U.S. Pat. No. 5,416,016; PCT Application WO 90/12096. Folic acid was conjugated to 3′-aminoalkyl-oligonucleotides at their 3′-terminus via carbodiimide chemistry. The multiplicity of folate receptors on membrane surfaces of most cells and the associated receptor mediated endocytotic processes were implicated in the enhanced transport of these oligonucleotide-folic acid conjugates into cells. There are however, several limitations to this approach for the conjugation of folic acid to oligonucleotides.
Folic acid and many related folates and antifolates exhibit very poor solubility that hinders the effective conjugation of folic acid to oligonucleotides and subsequent purification of oligonucleotide-folic acid conjugates. Further folic acid bears two reactive carboxylic acid groups that are just as likely to react with the terminal amino group of the 3-aminoalkyl-oligonucleotide. Thus conjugation will typically result in a mixture of a- and g-conjugates arising from the reaction of the a-carboxylate and the g-carboxylate of the glutamic acid portion of the folic acid molecule. This poses difficulties from the standpoint of characterizing the conjugate and further from the standpoint of polyglutamylation of folates. Polyglutamylation of folates is a well recognized phenomenon that has significant implications on the transport, localization and activity of folates. Since polyglutamylation rates differ significantly between the α- and γ-carboxylates, the use of poorly defined mixtures of oligonucleotide-folate conjugates, as obtained from the Low et al. procedure, U.S. Pat. No. 5,108,921, will lead to variable transport and concentration of the conjugate. Further, the conjugation of folates onto one end of an oligonucleotide may be a disadvantage because of the known propensity of exonucleases to rapidly cleave oligonucleotides by excising the terminal residues. Also, it has been observed that oligonucleotide-folic acid conjugates prepared in this fashion are light sensitive.
Therefore, there is a clear need for new oligonucleotide-folate conjugates, and methods for their preparation, that address the shortcomings of oligonucleotide conjugates as described above. The present invention is directed to this very important end.